The long term goal of this study is to better understand the physiologic mechanisms and chemical mediators responsible for opiate withdrawal. We have observed that a unique population of neurons located in the ventrolateral region of the midbrain periaqueductal gray (PAG) show a marked increase (approximately 500%) in pro-thyrotropin-releasing hormone (proTRH) gene expression during opiate withdrawal. Since excitation of this region evokes a quiescent and hyporeactive pattern of behavior, it is hypothesized that proTRH neurons in the ventrolateral PAG are activated as a compensatory response to the hyperactive state of opiate withdrawal. It is further hypothesized that one or more proTRH-derived peptides may mediate these responses. We propose to elucidate the anatomical connectivity of this unique population of opiate-responsive proTRH neurons in the ventrolateral PAG using confocal laser microscopy and electron microscopy as a way to identify specific pathways in the brain that are involved in opiate-withdrawal responses and determine how these neurons are integrated into the control system that responds to the hyperactive state of morphine withdrawal. In addition, we will determine whether the cell specific responses of these neurons to opiate withdrawal are due to the presence of opiate receptors on these cells and if CREB or upregulation of CREB-related proteins mediate these responses. Because these neurons are in a location where they could also be involved in the modulation of pain, an increase in proTRH gene activity in the PAG following deep noxious stimulation in continuously anesthetized animals will be determined. The role of proTRH-derived peptides in opiate withdrawal and antinociception will be further studied by inhibition of proTRH gene expression using adenovirus vectors containing an antisense proTRH transgene, stereotaxically injected into the PAG and their effect on behavioral and autonomic responses during withdrawal quantified. In parallel, will identify the proTRH-derived peptides in the PAG that increase during opiate withdrawal by chromatographic and electrophoretic techniques, and based on this analysis, peptides will be synthesized and injected into discrete regions of the brain to measure its effect on withdrawal responses. As heroin addiction continues to be on the common drugs of abuse, the data generated from this proposal could have clinical significance in the design of new approaches to the treatment of addiction and the withdrawal syndrome.